Fig. 1
Reduced free Ub levels and proteasome activity in neurons and brain slices treated with Aβ. (a) Cell viability detection with MTT assay. Primary neurons were isolated from mouse embryonic brains at 14.5 dpc and treated with Aβ40 or Aβ42 at DIV1 and analyzed by MTT assay at DIV5 (n = 3 per group). (b) Primary neurons were treated with Aβ40 or Aβ42 at DIV1 and stained for TUJ1 and CC3 at DIV5. DNA was visualized with DAPI. Scale bar, 100 μm (c) Gfap, Lcn2, and Tnf-α mRNA levels were determined by qRT-PCR in Aβ40- or Aβ42-treated primary neurons (n = 3 per group). The expression levels of Gfap, Lcn2, and Tnf-α were normalized to Gapdh levels and are expressed as the fold change relative to Aβ40-treated neurons. (d) Immunoblot detection of Ub (free, Ub1; conjugates, Ubn) and TUJ1 in Aβ-treated primary neurons. The neurons were treated with Aβ40 or Aβ42 at DIV1 and harvested at DIV5. TUJ1 was used as a neuronal marker and β-actin (β-Act) was used as a loading control (left). Band intensities of free Ub were calculated using ImageJ software, normalized to the loading control β-actin, and are expressed as the fold change relative to Aβ40-treated neurons (n = 3 per group) (right). (e) Aβ40- and Aβ42-treated primary neurons were harvested at DIV5 and subjected to a proteasome activity assay (n = 3 per group). Proteasome activities were calculated by subtracting the values of MG-132 treated samples from those of experimental samples and are expressed as the fold change relative to Aβ40-treated neurons. (f) Immunoblot detection of Ub, TUJ1, and SYN1 in Aβ-treated brain slice cultures. The slices were treated with Aβ40 or Aβ42 at DEV1 and harvested at DEV5. TUJ1 and SYN1 were used as neuronal markers and β-actin was used as a loading control (left). Band intensities of free Ub were calculated as above (n = 4 per group) (right). (g) Aβ40- and Aβ42-treated brain slices were subjected to a proteasome activity assay at DEV5 as above (n = 3 per group). All data are expressed as the means ± SEM from the indicated number of samples and representative images or immunoblot results are shown. #P < 0.1; *P < 0.05; ***P < 0.001 vs control